The invention relates to assays to detect tryptase activity and polypeptide tryptase substrates utilized in the assay.
Complete bibliographic citations to the references noted herein are included in the Bibliography section, immediately preceding the claims.
Mast cells are distributed on all epithelial and mucosal surfaces of the body. In addition to being found in mucous membranes of the respiratory and gastrointestinal tract, mast cells are also located near blood vessels in connective tissue. Mast cells play an important role in innate and acquired immune responses through the release of dense granules upon activation. The major component of mast cell secretory granules is serine proteases (Schwartz, L., et al.).
Human xcex2-tryptase is the most abundant and unique member of the serine protease family. Although xcex2-tryptase has uncertain physiological functions, it has been implicated as an effector in a plethora of human allergic and pathophysiological conditions, including asthma, arteriosclerosis, cancer, otitis media, arthritis, interstitial cystitis, rhinitis, dermatitis, and other deep organ diseases. Its prominent role in tissue remodeling and angiogenesis is suggestive of potentially beneficial physiological processes. There are at least three proteolytically active isoformns of tryptase present in mast cells, xcex2I-tryptase, xcex2II-tryptase, and xcex2III-tryptase. These tryptase isoforms are secreted as catalytically active tetramers (xcx9c135 kD) that are resistant to inactivation by plasma inhibitors.
The xcex2-tryptase enzyme has been recently crystallized, and the structure suggests that the association of the tryptase subunits into the native tetramer results in a stereospecific admission of potential substrates to the active site of each subunit. Although several in vitro studies have identified multiple substrates for tryptase, including neuropeptides, fibrinogen, stromelysin, pro-urokinase, prothrombin, and protease activated receptor-2, the physiologically relevant in vivo target of xcex2-tryptases""s serine protease activity has eluded discovery.
Human chromosome 16 encodes at least four homologous, yet distinct, tryptase genes, designated xcex1-, xcex2I-, xcex211-, and xcex2III-tryptase (Pallaoro, M., et al.). As used herein the unmodified term xe2x80x9ctryptasexe2x80x9d shall be used to refer to all tryptase isoforms. Two xcex2-tryptase isoforms share greater than 99% sequence identity, the xcex2I- and xcex2II-tryptases differing by only a single N-glycosylation site. It is not clear why so many highly similar tryptases are expressed by mast cells. One possibility is that they each perform different proteolytic functions that may be reflected in their substrate specificity preferences. Indeed, it has recently been shown that a single amino acid substitution between tryptase xcex1 and tryptase xcex2II accounts for discrimination in substrate preference for the two enzymes (Huang, C., et al.).
It has been difficult to study xcex2-tryptase and its physiological role because there are no suitable animal models for human allergies. Further, the human xcex2-tryptases show little or no homology with the tryptases found in animals other than primates. Finally, isolating natural xcex2-tryptase from human cadavers is a tedious and biohazardous undertaking. Only recently has recombinant, enzymatically-active tryptase become available through the work of the assignee of the present application, Promega Corporation of Madison, Wis., USA. (See co-pending and co-owned U.S. patent applications Ser. No. 09/598,982, filed Jun. 21, 2000, and Ser. No. 091079,970, filed 15 Apr. 1998, the entire contents of which are incorporated herein.)
During the past decade, clinicians have appreciated and reported the value of measuring released tryptase in making atopic diagnoses as well as when monitoring the course of mast cell-mediated disease. (xe2x80x9cAtopicxe2x80x9d being an umbrella term designating disease states characterized by symptoms produced upon exposure to an excitatory antigen or conditions such as asthma and other allergic reaction) xcex2-tryptase may be detected in the serum of non-atopic xe2x80x9cnormalxe2x80x9d individuals, and population serum levels are typically less than 1000 picograms of xcex2-tryptase per milliliter of serum. Conversely, serum tryptase levels are markedly raised in atopic subjects. Too often however, immunological detection (i.e., ELISA, RIA, PCFIA, and related assays of tryptase) is fraught with poor sensitivity or availability (e.g., the Schwartz ELISA method) and the requisite need for expensive ancillary detection equipment. Except in cases of exaggerated mast cell burden or degranulation, such as occurs during mastocytosis or anaphylaxis, it has been difficult to establish non-atopic or remission baselines of tryptase.
Conventional methods of assaying for tryptase proteolytic activity are hampered by poor specificity. These methods use substrates that are only intended for the measurement of xe2x80x9ctrypsin-likexe2x80x9d activity, particularly in purified tryptase preparations. For example, Benzoyl-Arg-paranitroaniline (trypsin), Tosyl-Gly-Pro-Arg-pNa (thrombin), Tosyl-Gly-Pro-Lys-pNa (plasmin), and Tosyl-Arg-Methyl-Ester exhibit cleavage upon contact by tryptase, but also are cleaved by other serine proteases. Because tryptase and related blood-borne serine proteases are able to cleave these substrates, they are of little value in ascertaining tryptase activity levels in complex biological samples.
The invention, which is defined by the claims set out at the end of this disclosure, is intended to solve at least some of the problems noted above.
A first embodiment of the invention is directed to an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, wherein P4 is Proline (xe2x80x9cPxe2x80x9d), P3 is Arginine (xe2x80x9cRxe2x80x9d) or Lysine (xe2x80x9cKxe2x80x9d), P2 is any amino acid, and P1 is K or R (SEQ. ID. NO: 1). For amino acid abbreviations, see Table 1 below. These isolated polypeptides function as very specific substrates that can be cleaved by the action of tryptases.
A second embodiment of the invention is directed to a method of assaying activity of an enzymatically-active xcex2-tryptase in a sample. The method comprises first contacting the sample with an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, where P4 is P, P3 is R or K, P2 is any amino acid, and xcex2I is K or R (SEQ. ID. NO: 1). The isolated polypeptide also includes a detectable leaving group bound to P4-P3-P2-P1, and is amino-terminally blocked. The sample is contacted with the isolated polypeptide under conditions wherein an amount of the detectable leaving group is cleaved from P4-P3-P2-P1 upon action of xcex2-tryptase present in the sample. The amount of detectable leaving group cleaved from the polypeptide is then quantified to give an indication of the extent of tryptase activity in the sample.
In the preferred embodiment of the method, the sample is contacted with an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, wherein P4 is acetylated, and wherein P4-P3-P2-P1 is selected from the group consisting of P-R-N-K (SEQ. ID. NO: 2), P-K-N-K (SEQ. ID. NO: 3), P-R-N-R (SEQ. ID. NO: 4), P-K-N-R (SEQ. ID. NO: 5), P-A-N-K (SEQ. ID. NO: 6), and P-R-T-K (SEQ. ID. NO: 7) (wherein asparagine is xe2x80x9cNxe2x80x9d and threonine is xe2x80x9cTxe2x80x9d), and further wherein a fluorogenic leaving group comprising 7-amino-4-carbamoylmethyl- coumarin is bound via an amide bond to P4-P3-P2-P1 at a carboxy-terminus of P4-P3-P2-P1. Here, if the sample has any tryptase activity, such activity will produce a detectable fluorescent moiety. The fluorescence of the sample is then measured to determine whether it undergoes a detectable change in fluorescence, the detectable change being an indication of the activity of the enzymatically-active xcex2-tryptase in the sample. The sample may be any sample suspected of containing tryptase activity, including whole blood, serum, plasma, urine, tears, lavage, tissue extract, conditioned media, etc.
A third embodiment of the invention is directed to a kit for analyzing samples for xcex2-tryptase activity. The kit comprises an isolated polypeptide comprising, in amino to carboxy order, P4-P3-P2-P1, wherein P4 is P, P3 is R or K, P2 is any amino acid, and P1 is K or R (SEQ. ID. NO: 1), and wherein a detectable leaving group is covalently bound to P4-P3-P2-P1, with the isolated polypeptide being disposed in a suitable container. The kit may also contain P4-P3-P2-P1 with a serine protease reactive moiety. The kit may optionally contain a supply of recombinant tryptase to be used to generate a standard curve, as well as a supply of aprotinin or a functional equivalent thereof. It is much preferred that instructions for use of the kit accompany each kit.
As noted above, mast cells express at least four distinct tryptase genes: xcex1, xcex2I, xcex2II, and xcex2III. It is currently unknown if these proteases perform the same or different functions. Based on the data presented herein, xcex2I, and xcex2II-tryptases have very similar P4 to P1 substrate preferences. This shared preference for peptide substrates likely extends to a shared preference for physiological substrates. Indeed, the optimal sequence for xcex2-tryptase, P4=P, P3=R or K, P2=any amino acid, and P1=K or R, is found in many of the macromolecular substrates that have been shown, at least in vitro, to be cleaved by tryptase, which preferentially cleaves after R or K.
For example, activation of the plasminogen cascade resulting in the destruction of extracellular matrix for cellular extravasation and migration may be a function of tryptase activation of pro-urokinase plasminogen activator at the P4-P1 sequence of Pro-Arg-Phe-Lys (SEQ. ID. NO: 8) (Stack, M., and Johnson, D.). Vasoactive intestinal peptide, a neuropeptide that is implicated in the regulation of vascular permeability, is cleaved by tryptase mainly after the arginines at the Thr-Arg-Leu-Arg (SEQ. ID. NO: 9) sequence (Stack, M., and Johnson, D.). The G-protein coupled receptor, PAR-2, can be cleaved and activated by tryptase at Ser-Lys-Gly-Arg (SEQ. ID. NO: 10), whereas the thrombin activated receptor, PAR-1, is inactivated by tryptase after the site Pro-Asn-Asp-Lys (SEQ. ID. NO: 11) (Jameson, G., et al.).
In the work leading to this invention, enzymatically-active xcex2I- and xcex2II-tryptases were heterologously expressed and purified in yeast to characterize the substrate specificity of each enzyme. Several positional scanning combinatorial tetrapeptide substrate libraries were used to dissect the primary and extended substrate specificity. Both enzymes have a strict primary preference for cleavage after the basic amino acids lysine and arginine, with only a slight preference for lysine over arginine. xcex2I- and xcex2II-tryptases share similar extended substrate specificity, preferring to cleave after P4-proline, P3-arginine or lysine, with P2 having some asparagine and threonine selectivity (N-preference, FIG. 3A).
It is shown herein that xcex2I- and xcex2II-tryptases have a defined primary substrate specificity (i.e., the residue at the P1 position) and a defined extended substrate specificity (i.e., the residues at the P4-P2 positions). The library profiles generated in developing the invention described and claimed herein indicate that the substrate specificity is similar for the two enzymes. Purthermore, single substrates were designed and assayed to test the extended substrate specificity requirements, thereby yielding sensitive and selective substrates for xcex2-tryptases. Structural determinants of specificity were examined through the modeling of the optimized substrate into the active site of the tryptase structure. Finally, the specificity determined in this study correlates with the cleavage sites found in many of the characterized physiological substrates and may lead to the identification of additional substrates in both the immunity and pathology of xcex2I- and xcex2II-tryptases.
The invention described herein highlights the utility of using generalized positional scanning combinatorial peptide libraries to characterize functional similarities and differences between homologous enzymes, to generate sensitive and selective tryptase substrates and inhibitors, and to define a subset of potential physiological tryptase substrates.
The objects and advantages of the invention will appear more fully from the following detailed description of the preferred embodiment of the invention made in conjunction with the accompanying drawings.